The research activities described in this application concern the elucidation of regulatory mechanisms governing the three mitochondrial alpha-keto acid dehydrogenase multienzyme complexes (e.g., pyruvate, alpha-ketoglutarate and branched chain alpha-keton acid) in the heart. These important enzyme complexes individually catalyze the oxidative decarboxylation of the individual alpha-keto acids with the resultant production of CO2, NADA and the appropriate acyl-CoA derivative. A major emphasis of the proposed research will be the investigation of the regulation of the pyruvate dehydrogenase complex (PDC) as we have developed considerable experience manipulating this enzyme complex in intact metabolic systems. PDC is subject to numerous well substantiated regulatory factors including direct effects on the active enzyme complex and covalent modification (i.e., phosphorylation) by a protein kinase/phosphatase system which interconverts an active and inactive form of the enzyme. The regulation of the various enzyme complexes will be pursued primarily in metabolic systems derived from the heart and liver. For reasons of comparison occasional experiments will be performed using metabolic preparations from kidney, skeletal muscle and brain. The regulation of the alpha-keto acid dehydrogenases will be studied at the level of the isolated perfused organ, the isolated mitochondiron, and the purified enzyme complex. Simultaneous measurement of metabolic flux through the complex, the activity state of extractable enzyme complex and the concentrations of various tissue metabolites and nucleotides will be performed in order to characterize the various factors affecting the regulation of these enzyme complexes. These parameters will be monitored during metabolic state changes in the tissues under study including alterations in the composition and concentration of various substrates, hormonal treatment and various compounds or drugs which either activate or inhibit the metabolic flux through these reactions. It is anticipated that the results of the proposed research activities will provide a bridge between the purely enzymological and the wholely physiological approaches to the metabolic regulation of three enzyme complexes which are primary in the processes of carbon movement and energy generation in cardiac as well as other tissues.